Methods for diagnosis and prognosis of epithelial cancers

ABSTRACT

The present invention is based on the discovery that three proteins, Cystatin B, Chaperonin 10, and Profilin are present in the urine of patients with bladder cancer, a cancer of epithelial origin. Accordingly, the present invention is directed to methods for prognostic evaluation of cancers of epithelial origin and to methods for facilitating diagnosis of cancers of epithelial origin by monitoring the presence of these markers in biological samples. The invention is also directed to markers for therapeutic efficacy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.provisional Patent Application No. 60/648,110 filed Jan. 28, 2005.

GOVERNMENT SUPPORT

This work was supported by National Institute of Health grant number2R37 CA37393. The government has certain rights to the invention.

BACKGROUND OF THE INVENTION

One of the most important factors in the survival of cancer is detectionat an early stage. Clinical assays that detect the early events ofcancer offer an opportunity to intervene and prevent cancer progression.With the development of gene profiling and proteomics there has beensignificant progress in the identification of molecular markers or“biomarkers” that can be used to diagnose and prognose specific cancers.For example, in the case of prostate cancer, the antigen PSA (forprostate specific antigen) can be detected in the blood and isindicative of the presence of prostate cancer. Thus, the blood of men atrisk for prostate cancer can be quickly, easily, and safely screened forelevated PSA levels.

Even though there has been significant progress in the field of cancerdetection, there still remains a need in the art for the identificationof new biomarkers for a variety of cancers that can be easily used inclinical applications. For example, to date there are relatively fewoptions available for the diagnosis of breast cancer using easilydetectable biomarkers. Overexpression of EGFR, particularly coupled withdown-regulation of the estrogen receptor, is a marker of poor prognosisin breast cancer patients. Other known markers of breast cancer includehigh levels of M2 pyruvate kinase (M2 PK) in blood (U.S. Pat. No.6,358,683), high ZNF217 protein levels in blood (WO 98/02539), anddifferential expression of a newly identified protein in breast cancer,PDEBC, which is useful for diagnosis (U.S. patent application No.20030124543). Cell surface markers such as CEA, CA-125 and HCG arefrequently elevated in the serum of patients with locally advanced andmetastatic bladder cancer (Izes et al., J Urol. June; 165(6 Pt1):1908-13, 2001), and studies involving circulating levels oftumor-related proteins such as matrix metalloproteinase-2 (Gohji et al.,Cancer Research 56:3196, 1996), hepatocyte growth factor (Gohji et al.,J. Clin. Oncol. 18:2963, 2000), and tissue polypeptide antigen(Maulard-Durdux et al., J. Clin. Oncol. 15:3446, 1997) have shownpromise. These biomarkers offer alternative methods of diagnosis,however, they are not widely used. Furthermore, despite the use of anumber of histochemical, genetic, and immunological markers, cliniciansstill have a difficult time predicting which tumors will metastasize toother organs.

The identification of cancer biomarkers is particularly relevant toimproving diagnosis, prognosis, and treatment of the disease. As such,there is need in the art to identify alternative biomarkers that can bequickly, easily, and safely detected. Such biomarkers may be used todiagnose, to stage, or to monitor the progression or treatment of asubject with bladder cancer, in particular, an invasive, potentiallymetastatic stage of the disease.

SUMMARY OF THE INVENTION

The present invention is based on the surprising discovery that threeproteins, Cystatin B, Chaperonin 10, and Profilin (also referred to as“epithelial cancer markers”), are present in the urine of patients withbladder cancer, a cancer of epithelial origin. Accordingly, the presentinvention is directed to methods for prognostic evaluation of cancers ofepithelial origin and to methods for facilitating diagnosis of cancersof epithelial origin by monitoring the presence of these markers inbiological samples. The invention is also directed to markers fortherapeutic efficacy. In particular, the amount of Cystatin B detectedin urine correlates with disease status such that Cystatin B levels canbe used to predict the presence of invasive bladder cancer. Thus,measuring the level of Cystatin B, Chaperonin 10, and/or Profilinproteins in urine provides a quick, easy, and safe screen that can beused to both diagnose and prognose bladder cancer in a patient.Alternatively, the absence of these markers can provide an indicationthat the patient does not have bladder cancer.

In one embodiment, a method for facilitating the diagnosis of cancer ofan epithelial origin in a patient is provided. The method comprisesobtaining a biological sample, preferably a voided urine specimen, froma patient and detecting the presence or absence of at least oneepithelial cancer biomarker (Cystatin B, Chaperonin 10, or Profilin) inthe sample, wherein the presence of at least one epithelial cancerbiomarker is indicative of cancer of epithelial origin.

Biological samples, for example, can be obtained from blood, tissue(e.g. tumor or breast), serum, stool, urine, sputum, cerebrospinalfluid, nipple aspirates and supernatant from cell lysate. One preferredbiological sample is urine.

As used herein, “cancer of epithelial origin” refers to cancers thatarise from epithelial cells which include, but are not limited to,breast cancer, basal cell carcinoma, adenocarcinoma, gastrointestinalcancer, lip cancer, mouth cancer, esophageal cancer, small bowel cancerand stomach cancer, colon cancer, liver cancer, bladder cancer, pancreascancer, ovary cancer, cervical cancer, lung cancer, breast cancer andskin cancer, such as squamous cell and basal cell cancers, prostatecancer, renal cell carcinoma, and other known cancers that effectepithelial cells throughout the body.

In one embodiment, a method for facilitating the diagnosis of bladdercancer in a patient is provided. The method comprises obtaining abiological sample, preferably a voided urine specimen, from a patientand detecting the presence or absence of at least one epithelial cancerbiomarker (Cystatin B, Chaperonin 10, or Profilin) in urine sample,wherein the presence of at least one epithelial cancer biomarker isindicative of bladder cancer.

In another embodiment, the method for diagnosing a cancer of epithelialorigin is provided. The comprises measuring the level of at least oneepithelial cancer biomarker present in a biological sample (test sample)from a patient and comparing the observed level of at least one marker(Cystatin B, Chaperonin 10, or Profilin) with the level of the markerpresent in a control sample of the same type. Higher levels of markersin the test sample, as compared to the control sample, is indicative ofcancer of epithelial origin.

In one preferred embodiment, the methods of the invention are used forearly detection of cancer. For example, a patient can be screened by aphysician during their physical.

In one embodiment, a method for diagnosing bladder cancer is provided.The method comprises measuring the level of at least one epithelialcancer biomarker (Cystatin B, Chaperonin 10, or Profilin) present in abiological sample (the test sample) from a patient and comparing theobserved level of at least one marker with the level of the markerpresent in a control sample of the same type. Higher levels of markersin the test sample, as compared to the control sample, is indicative ofbladder cancer.

In one embodiment, a method for diagnosing invasive bladder cancer in apatient is provided. The method comprises measuring levels of Cystatin Bepithelial cancer biomarker present in a biological sample obtained fromthe patient (test sample) and comparing the level of Cystatin B in thetest sample with the level of Cystatin B present in a non-invasivecancer control sample. A higher level of Cystatin B in the test sampleas compared to the level of Cystatin B in the control sample isindicative of invasive bladder cancer.

The term “control sample” refers to a biological sample (e.g. blood,urine, tumor) obtained from a “normal” or “healthy” individual(s) thatis believed not to have cancer. Controls may be selected using methodsthat are well known in the art. Once a level has become well establishedfor a control population, array results from test biological samples canbe directly compared with the known levels.

The term “non-invasive control sample” refers to a biological sampleobtained from a individual(s) that has a non-invasive form of cancer.Once a level has become well established for a control population, arrayresults from test biological samples can be directly compared with theknown levels.

The term “test sample” refers to a biological sample obtained from apatient being tested for a cancer of epithelial origin.

The present invention also contemplates the assessment of the level ofepithelial cancer biomarker present in multiple test samples obtainedfrom the same patient, where a progressive increase in the amount of themarker over time indicates an increased aggressiveness (e.g. metastaticpotential) of the cancer tumor. As such, the levels of the epithelialcancer biomarker serve as a predictor of disease status and stage.

The present invention further contemplates the assessment of epithelialcancer biomarker/s to monitor the therapeutic efficacy of a treatmentregime designed to treat a patient having a cancer of epithelial origin(e.g. bladder cancer).

In one aspect of the invention, epithelial cancer biomarker levels (e.g.(Cystatin B, Chaperonin 10, or Profilin) present in a test biologicalsample are measured by contacting the test sample, or preparationthereof, with an antibody-based binding moiety that specifically bindsto the epithelial cancer biomarker, or to a portion thereof.

Antibody-based immunoassays are the preferred means for measuring levelsof biomarkers. However, any means known to those skilled in art can beused to assess biomarker levels. For example, biomarker levels can beassessed by mass spectrometry, including SELDI mass spectrometry.

In a further embodiment, the invention provides for kits that comprisemeans for measuring at least one epithelial cancer biomarker in abiological sample. The kit comprises a container for holding abiological sample (e.g. urine sample), and at least one antibody thatspecifically binds an epithelial cancer biomarker.

In one embodiment, the kit provides comprises two antibodies thatspecifically binds to an epithelial cancer biomarker. In one embodiment,one antibody is immobilized on a solid phase and one antibody isdetectably labeled. The kits can comprise can comprise anti-Cystatin B,anti-Chaperonin 10, and/or anti-Profilin antibodies.

Other aspects of the invention are disclosed infra.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate embodiments of the invention and,together with the description, serve to explain the objects, advantages,and principles of the invention.

FIG. 1 is a flow diagram showing the approach to epithelial cancerbiomarker discovery.

FIG. 2 shows comparative 2D page of invasive bladder tumor tissue andnormal bladder tissue reveals many potential spots. The circled spot wasidentified by mass spectroscopy to be Cystatin B.

FIG. 3 shows a graph depicting the results of a semi-quantitativeWestern Blot analysis of Cystatin B detected in voided urine specimens.

DETAILED DESCRIPTION OF THE INVENTION

We have discovered that three proteins, Cystatin B, Chaperonin 10, andProfilin (referred to herein as “epithelial cancer markers”), arepresent in the urine of patients that have cancers of epithelial origin.Levels of Cystatin B present in urine samples of patients correlate withthe presence of bladder cancers, in particular invasive bladder cancers.

The term “aggressive” or “invasive” with respect to cancer refers to theproclivity of a tumor for expanding beyond its boundaries into adjacenttissue (Darnell, J. (1990), Molecular Cell Biology, Third Ed., W. H.Freeman, NY). Invasive cancer can be contrasted with organ-confinedcancer wherein the tumor is confined to a particular organ. The invasiveproperty of a tumor is often accompanied by the elaboration ofproteolytic enzymes, such as collagenases, that degrade matrix materialand basement membrane material to enable the tumor to expand beyond theconfines of the capsule, and beyond confines of the particular tissue inwhich that tumor is located. Invasive bladder cancer includes invasiveinto Muscularis Propria and/or Lamina Propria.

The term “metastasis”, as used herein, refers to the condition of spreadof cancer from the organ of origin to additional distal sites in thepatient. The process of tumor metastasis is a multistage event involvinglocal invasion and destruction of intercellular matrix, intravasationinto blood vessels, lymphatics or other channels of transport, survivalin the circulation, extravasation out of the vessels in the secondarysite and growth in the new location (Fidler, et al., Adv. Cancer Res.28, 149-250 (1978), Liotta, et al., Cancer Treatment Res. 40, 223-238(1988), Nicolson, Biochim. Biophy. Acta 948, 175-224 (1988) and Zetter,N. Eng. J. Med. 322, 605-612 (1990)). Increased malignant cell motilityhas been associated with enhanced metastatic potential in animal as wellas human tumors (Hosaka, et al., Gann 69, 273-276 (1978) and Haemmerlin,et al., Int. J. Cancer 27, 603-610 (1981)).

As used herein, a “biological sample” refers to a urine sample obtainedfrom a patient. Biological samples, for example, can be obtained fromblood, tissue (e.g. tumor or breast), serum, stool, urine, sputum,cerebrospinal fluid, nipple aspirates and supernatant from cell lysate.One preferred biological sample is urine.

In a preferred embodiment, the biological sample is treated as toprevent degradation of epithelial cancer biomarkers. Methods forinhibiting or preventing degradation include, but are not limited to,treatment of the sample with protease, freezing the sample, or placingthe sample on ice. Preferably, prior to analysis, the samples areconstantly kept under conditions as to prevent degradation of themarkers.

As used herein, a “tumor sample” refers to a portion, piece, part,segment, or fraction of a tumor, for example, a tumor which is obtainedor removed from a subject (e. g., removed or extracted from a tissue ofa subject), preferably a human subject.

As used herein, Cystatin B refers to the protein of Genebank accessionNM_000100.2, NP_000091 (Homosapiens). The term also encompasses speciesvariants, homologues, allelic forms, mutant forms, and equivalentsthereof.

As used herein, Chaperonin 10 refers to the protein of Genebankaccession, protein, AAA50953 (Homosapiens). The term also encompassesspecies variants, homologues, allelic forms, mutant forms, andequivalents thereof.

As used herein, Profilin refers to the protein of Genebank accession,protein, A28622 (Homosapiens). The term also encompasses speciesvariants, homologues, allelic forms, mutant forms, and equivalentsthereof.

The present invention is directed to methods for facilitating diagnosisof cancers of epithelial origin in a patient. In one embodiment, themethod comprises obtaining a biological sample from a patient anddetecting the presence or absence of at least one epithelial cancerbiomarker (Cystatin B, Chaperonin 10, or Profilin) in the sample,wherein the presence of at least one marker is indicative of thepresence of cancer of epithelial origin.

In another embodiment, the methods involve measuring levels of at leastone epithelial cancer biomarker (Cystatin B, Chaperonin 10, or Profilin)in a test sample obtained from a patient being tested for cancer, andcomparing the observed levels to the levels of the epithelial cancerbiomarker found in a control sample, for example a sample obtained froman individual patient or population of individuals that do not to havecancer. Levels of at least one epithelial cancer biomarker higher thanlevels that are observed in the normal control indicate the presence ofcancer of epithelial origin. The levels of biomarkers can be representedby arbitrary units, for example as units obtained from a densitometer,luminometer, or an Elisa plate reader.

As used herein, “a higher level of at least one epithelial cancerbiomarker in the test sample as compared to the level in the controlsample” refers to an amount of at least one biomarker that is greaterthan an amount of the same biomarker present in a control sample. Theterm “higher level” refers to a level that is statistically significantor significantly above levels found in the control sample. The “higherlevel” can be for example 1.2 fold to 1.9 fold higher. Preferably, the“higher level” is at least 2 fold greater, or even 3 fold greater.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) above normal, or higher, concentration of the marker.

For purposes of comparison, the test sample and control sample are ofthe same type, that is, obtained from the same biological source. Thecontrol sample can also be a standard sample that contains the sameconcentration of the epithelial cancer biomarker that is normally foundin a biological sample that is obtained from a healthy individual.

In one aspect of the invention, a secondary diagnostic step can beperformed. For example, if a level of at least one epithelial cancerbiomarker is found to indicate the presence of cancer, then anadditional method of detecting the cancer can be performed to confirmthe presence of the cancer. Any of a variety of additional diagnosticsteps can be used, such as ultrasound, PET scanning, MRI, or any otherimaging techniques, biopsy, clinical examination, ductogram, or anyother method.

The present invention further provides for methods of prognosticevaluation of a patient suspected of having, or having, cancer ofepithelial origin. The method comprises measuring the level of at leastone epithelial cancer biomarker (Cystatin B, Chaperonin 10, or Profilin)present in a test biological sample obtained from a patient andcomparing the observed level with a range of at least one epithelialcancer biomarker levels normally found in biological samples (of thesame type) of healthy individuals. A high level for example, isindicative of a greater potential for metastatic activity andcorresponds to a poor prognosis, while lower levels indicate that thetumor is less aggressive and correspond to a better prognosis.

Additionally, disease progression can be assessed by following thelevels of at least one epithelial cancer biomarker in an individualpatient. For example, changes in the patients condition can be monitoredby comparing changes expression levels of Cystatin B, Chaperonin 10, orProfilin in the patient over time. Progressive increases in the levelsof at least one epithelial cancer biomarker is indicative of increasedpotential for tumor invasion and metastasis.

The prognostic methods of the invention also are useful for determininga proper course of treatment for a patient having cancer. A course oftreatment refers to the therapeutic measures taken for a patient afterdiagnosis or after treatment for cancer. For example, a determination ofthe likelihood for cancer recurrence, spread, or patient survival, canassist in determining whether a more conservative or more radicalapproach to therapy should be taken, or whether treatment modalitiesshould be combined. For example, when cancer recurrence is likely, itcan be advantageous to precede or follow surgical treatment withchemotherapy, radiation, immunotherapy, biological modifier therapy,gene therapy, vaccines, and the like, or adjust the span of time duringwhich the patient is treated.

The methods of the invention are suitable to diagnose or prognose anycancer of epithelial origin, including but not limited to, breastcancer, basal cell carcinoma, adenocarcinoma, gastrointestinal cancer,lip cancer, mouth cancer, esophageal cancer, small bowel cancer andstomach cancer, colon cancer, liver cancer, bladder cancer, pancreascancer, ovary cancer, cervical cancer, lung cancer, breast cancer andskin cancer, such as squamous cell and basal cell cancers, prostatecancer, renal cell carcinoma, and other known cancers that effectepithelial cells throughout the body.

In one preferred embodiment, the cancer of epithelial origin is bladdercancer.

Measuring Levels of at Least One Epithelial Cancer Biomarker

The levels of at least one epithelial cancer biomarker, as describedherein, can be measured by any means known to those skilled in the art.In the present invention, it is generally preferred to use antibodies,or antibody equivalents, to detect levels of at least one epithelialcancer biomarker protein in biological samples.

In one embodiment, levels of at least one epithelial cancer biomarkerprotein are measured by contacting the biological sample with anantibody-based binding moiety that specifically binds to at least oneepithelial cancer biomarker, or to a fragment of at least one epithelialcancer biomarker. Formation of the antibody-epithelial cancer biomarkercomplex is then detected as a measure of the epithelial cancer biomarkerlevels.

The term “antibody-based binding moiety” or “antibody” includesimmunoglobulin molecules and immunologically active determinants ofimmunoglobulin molecules, e.g., molecules that contain an antigenbinding site which specifically binds (immunoreacts with) to theepithelial cancer biomarker to be detected, e.g. Cystatin B, Chaperonin10, or Profilin. The term “antibody-based binding moiety” is intended toinclude whole antibodies, e.g., of any isotype (IgG, IgA, IgM, IgE,etc), and includes fragments thereof which are also specificallyreactive the epithelial cancer biomarker protein. Antibodies can befragmented using conventional techniques. Thus, the term includessegments of proteolytically-cleaved or recombinantly-prepared portionsof an antibody molecule that are capable of selectively reacting with acertain protein. Non limiting examples of such proteolytic and/orrecombinant fragments include Fab, F(ab′)2, Fab′, Fv, dAbs and singlechain antibodies (scFv) containing a VL and VH domain joined by apeptide linker. The scFv's may be covalently or non-covalently linked toform antibodies having two or more binding sites. Thus, “antibody-basebinding moiety” includes polyclonal, monoclonal, or other purifiedpreparations of antibodies and recombinant antibodies. The term“antibody-base binding moiety” is further intended to include humanizedantibodies, bispecific antibodies, and chimeric molecules having atleast one antigen binding determinant derived from an antibody molecule.In a preferred embodiment, the antibody-based binding moiety detectablylabeled.

“Labeled antibody”, as used herein, includes antibodies that are labeledby a detectable means and include, but are not limited to, antibodiesthat are enzymatically, radioactively, fluorescently, andchemiluminescently labeled. Antibodies can also be labeled with adetectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, or HIS.

In the diagnostic and prognostic methods of the invention that useantibody based binding moieties for the detection of at least oneepithelial cancer biomarker, the level of at least one epithelial cancerbiomarker present in the biological samples correlate to the intensityof the signal emitted from the detectably labeled antibody.

In one preferred embodiment, the antibody-based binding moiety isdetectably labeled by linking the antibody to an enzyme. The enzyme, inturn, when exposed to it's substrate, will react with the substrate insuch a manner as to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorometric or by visual means. Enzymeswhich can be used to detectably label the antibodies of the presentinvention include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-V-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-VI-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. Chemiluminescence is another method that can beused to detect an antibody-based binding moiety.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling an antibody, it ispossible to detect the antibody through the use of radioimmune assays.The radioactive isotope can be detected by such means as the use of agamma counter or a scintillation counter or by audoradiography. Isotopeswhich are particularly useful for the purpose of the present inventionare ³H, ¹³¹I, ³⁵S, ¹⁴C, and preferably ¹²⁵I.

It is also possible to label an antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are CYE dyes, fluorescein isothiocyanate, rhodamine,phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine.

An antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

An antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-antibodyis then determined by detecting the presence of luminescence that arisesduring the course of a chemical reaction. Examples of particularlyuseful chemiluminescent labeling compounds are luminol, luciferin,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

As mentioned above, levels of at least one epithelial cancer biomarkerprotein can be detected by immunoassays, such as enzyme linkedimmunoabsorbant assay (ELISA), radioimmunoassay (RIA), Immunoradiometricassay (IRMA), Western blotting, or immunohistochemistry, each of whichare described in more detail below. Immunoassays such as ELISA or RIA,which can be extremely rapid, are more generally preferred. Antibodyarrays or protein chips can also be employed, see for example U.S.Patent Application Nos: 20030013208A1; 20020155493A1; 20030017515 andU.S. Pat. Nos. 6,329,209; 6,365,418, which are herein incorporated byreference in their entirety.

Immunoassays

“Radioimmunoassay” is a technique for detecting and measuring theconcentration of an antigen, biomarker to be detected, using a labeled(e.g. radioactively labeled) form of the antigen. Examples ofradioactive labels for antigens include ³H, ¹⁴C, and ¹²⁵I. Theconcentration of antigen in a biological sample is measured by havingthe antigen in the biological sample compete with the labeled (e.g.radioactively) antigen for binding to an antibody that specificallybinds the antigen. To ensure competitive binding between the labeledantigen and the unlabeled antigen, the labeled antigen is present in aconcentration sufficient to saturate the binding sites of the antibody.The higher the concentration of antigen in the sample, the lower theconcentration of labeled antigen that will bind to the antibody.

In a radioimmunoassay, to determine the concentration of labeled antigenbound to antibody, the antigen-antibody complex must be separated fromthe free antigen. One method for separating the antigen-antibody complexfrom the free antigen is by precipitating the antigen-antibody complexwith an anti-isotype antiserum. Another method for separating theantigen-antibody complex from the free antigen is by precipitating theantigen-antibody complex with formalin-killed S. aureus. Yet anothermethod for separating the antigen-antibody complex from the free antigenis by performing a “solid-phase radioimmunoassay” where the antibody islinked (e.g., covalently) to Sepharose beads, polystyrene wells,polyvinylchloride wells, or microtiter wells. By comparing theconcentration of labeled antigen bound to antibody to a standard curvebased on samples having a known concentration of antigen, theconcentration of antigen in the biological sample can be determined.

A “Immunoradiometric assay” (IRMA) is an immunoassay in which theantibody reagent is radioactively labeled. An IRMA requires theproduction of a multivalent antigen conjugate, by techniques such asconjugation to a protein e.g., rabbit serum albumin (RSA). Themultivalent antigen conjugate must have at least 2 antigen residues permolecule and the antigen residues must be of sufficient distance apartto allow binding by at least two antibodies to the antigen. For example,in an IRMA the multivalent antigen conjugate can be attached to a solidsurface such as a plastic sphere. Unlabeled “sample” antigen andantibody to antigen which is radioactively labeled are added to a testtube containing the multivalent antigen conjugate coated sphere. Theantigen in the sample competes with the multivalent antigen conjugatefor antigen antibody binding sites. After an appropriate incubationperiod, the unbound reactants are removed by washing and the amount ofradioactivity on the solid phase is determined. The amount of boundradioactive antibody is inversely proportional to the concentration ofantigen in the sample.

The most common enzyme immunoassay is the “Enzyme-Linked ImmunosorbentAssay (ELISA).” ELISA is a technique for detecting and measuring theconcentration of an antigen using a labeled (e.g. enzyme linked) form ofthe antibody. There are different forms of ELISA, which are well knownto those skilled in the art. The standard techniques known in the artfor ELISA are described in “Methods in Immunodiagnosis”, 2nd Edition,Rose and Bigazzi, eds. John Wiley & Sons, 1980; Campbell et al.,“Methods and Immunology”, W. A. Benjamin, Inc., 1964; and Oellerich, M.1984, J. Clin. Chem. Clin. Biochem., 22:895-904.

In a “sandwich ELISA”, an antibody (e.g. anti-cystatin B,anti-chaperonin 10, or anti-profilin) is linked to a solid phase (i.e. amicrotiter plate) and exposed to a biological sample containing antigen(e.g. cystatin B, chaperonin 10, and/or profilin). The solid phase isthen washed to remove unbound antigen. A labeled antibody (e.g. enzymelinked) is then bound to the bound-antigen (if present) forming anantibody-antigen-antibody sandwich. Examples of enzymes that can belinked to the antibody are alkaline phosphatase, horseradish peroxidase,luciferase, urease, and B-galactosidase. The enzyme linked antibodyreacts with a substrate to generate a colored reaction product that canbe measured.

In a “competitive ELISA”, antibody is incubated with a sample containingantigen (i.e. at least one epithelial cancer biomarker). Theantigen-antibody mixture is then contacted with a solid phase (e.g. amicrotiter plate) that is coated with antigen (i.e., at least oneepithelial cancer biomarker). The more antigen present in the sample,the less free antibody that will be available to bind to the solidphase. A labeled (e.g., enzyme linked) secondary antibody is then addedto the solid phase to determine the amount of primary antibody bound tothe solid phase.

In a “immunohistochemistry assay” a section of tissue is tested forspecific proteins by exposing the tissue to antibodies that are specificfor the protein that is being assayed. The antibodies are thenvisualized by any of a number of methods to determine the presence andamount of the protein present. Examples of methods used to visualizeantibodies are, for example, through enzymes linked to the antibodies(e.g., luciferase, alkaline phosphatase, horseradish peroxidase, or.beta.-galactosidase), or chemical methods (e.g., DAB/Substratechromagen). It is also contemplated that tissue microarrays can be usedin methods of the invention.

Other techniques may be used to detect at least one epithelial cancerbiomarker, according to a practitioner's preference, based upon thepresent disclosure. One such technique is Western blotting (Towbin etat., Proc. Nat. Acad. Sci. 76:4350 (1979)), wherein a suitably treatedsample is run on an SDS-PAGE gel before being transferred to a solidsupport, such as a nitrocellulose filter. Detectably labeledanti-biomarker antibodies can then be used to assess the levels of atleast one epithelial cancer biomarker, where the intensity of the signalfrom the detectable label corresponds to the amount biomarker present.Levels can be quantitated, for example by densitometry.

Mass Spectometry

In addition, at least one epithelial cancer biomarker may be detectedusing Mass Spectrometry such as MALDI/TOF (time-of-flight), SELDI/TOF,liquid chromatography-mass spectrometry (LC-MS), gas chromatography-massspectrometry (GC-MS), high performance liquid chromatography-massspectrometry (HPLC-MS), capillary electrophoresis-mass spectrometry,nuclear magnetic resonance spectrometry, or tandem mass spectrometry(e.g., MS/MS, MS/MS/MS, ESI-MS/MS, etc.). See for example, U.S. PatentApplication Nos: 20030199001, 20030134304, 20030077616, which are hereinincorporated by reference.

Mass spectrometry methods are well known in the art and have been usedto quantify and/or identify biomolecules, such as proteins (see, e.g.,Li et al. (2000) Tibtech 18:151-160; Rowley et al. (2000) Methods 20:383-397; and Kuster and Mann (1998) Curr. Opin. Structural Biol. 8:393-400). Further, mass spectrometric techniques have been developedthat permit at least partial de novo sequencing of isolated proteins.Chait et al., Science 262:89-92 (1993); Keough et al., Proc. Natl. Acad.Sci. USA. 96:7131-6 (1999); reviewed in Bergman, EXS 88:133-44 (2000).

In certain embodiments, a gas phase ion spectrophotometer is used. Inother embodiments, laser-desorption/ionization mass spectrometry is usedto analyze the sample. Modern laser desorption/ionization massspectrometry (“LDI-MS”) can be practiced in two main variations: matrixassisted laser desorption/ionization (“MALDI”) mass spectrometry andsurface-enhanced laser desorption/ionization (“SELDI”). In MALDI, theanalyte is mixed with a solution containing a matrix, and a drop of theliquid is placed on the surface of a substrate. The matrix solution thenco-crystallizes with the biological molecules. The substrate is insertedinto the mass spectrometer. Laser energy is directed to the substratesurface where it desorbs and ionizes the biological molecules withoutsignificantly fragmenting them. However, MALDI has limitations as ananalytical tool. It does not provide means for fractionating the sample,and the matrix material can interfere with detection, especially for lowmolecular weight analytes. See, e.g., U.S. Pat. No. 5,118,937(Hillenkamp et al.), and U.S. Pat. No. 5,045,694 (Beavis & Chait).

In SELDI, the substrate surface is modified so that it is an activeparticipant in the desorption process. In one variant, the surface isderivatized with adsorbent and/or capture reagents that selectively bindthe protein of interest. In another variant, the surface is derivatizedwith energy absorbing molecules that are not desorbed when struck withthe laser. In another variant, the surface is derivatized with moleculesthat bind the protein of interest and that contain a photolytic bondthat is broken upon application of the laser. In each of these methods,the derivatizing agent generally is localized to a specific location onthe substrate surface where the sample is applied. See, e.g., U.S. Pat.No. 5,719,060 and WO 98/59361. The two methods can be combined by, forexample, using a SELDI affinity surface to capture an analyte and addingmatrix-containing liquid to the captured analyte to provide the energyabsorbing material.

For additional information regarding mass spectrometers, see, e.g.,Principles of Instrumental Analysis, 3rd edition., Skoog, SaundersCollege Publishing, Philadelphia, 1985; and Kirk-Othmer Encyclopedia ofChemical Technology, 4.sup.th ed. Vol. 15 (John Wiley & Sons, New York1995), pp. 1071-1094.

Detection of the presence of a marker will typically involve detectionof signal intensity. This, in turn, can reflect the quantity andcharacter of a polypeptide bound to the substrate. For example, incertain embodiments, the signal strength of peak values from spectra ofa first sample and a second sample can be compared (e.g., visually, bycomputer analysis etc.), to determine the relative amounts of particularbiomolecules. Software programs such as the Biomarker Wizard program(Ciphergen Biosystems, Inc., Fremont, Calif.) can be used to aid inanalyzing mass spectra. The mass spectrometers and their techniques arewell known to those of skill in the art.

Any person skilled in the art understands, any of the components of amass spectrometer (e.g., desorption source, mass analyzer, detect, etc.)and varied sample preparations can be combined with other suitablecomponents or preparations described herein, or to those known in theart. For example, in some embodiments a control sample may contain heavyatoms (e.g. ¹³C) thereby permitting the test sample to mixed with theknown control sample in the same mass spectrometry run.

In one preferred embodiment, a laser desorption time-of-flight (TOF)mass spectrometer is used. In laser desorption mass spectrometry, asubstrate with a bound marker is introduced into an inlet system. Themarker is desorbed and ionized into the gas phase by laser from theionization source. The ions generated are collected by an ion opticassembly, and then in a time-of-flight mass analyzer, ions areaccelerated through a short high voltage field and let drift into a highvacuum chamber. At the far end of the high vacuum chamber, theaccelerated ions strike a sensitive detector surface at a differenttime. Since the time-of-flight is a function of the mass of the ions,the elapsed time between ion formation and ion detector impact can beused to identify the presence or absence of molecules of specific massto charge ratio.

In some embodiments the relative amounts of one or more biomoleculespresent in a first or second sample is determined, in part, by executingan algorithm with a programmable digital computer. The algorithmidentifies at least one peak value in the first mass spectrum and thesecond mass spectrum. The algorithm then compares the signal strength ofthe peak value of the first mass spectrum to the signal strength of thepeak value of the second mass spectrum of the mass spectrum. Therelative signal strengths are an indication of the amount of thebiomolecule that is present in the first and second samples. A standardcontaining a known amount of a biomolecule can be analyzed as the secondsample to provide better quantify the amount of the biomolecule presentin the first sample. In certain embodiments, the identity of thebiomolecules in the first and second sample can also be determined.

In one preferred embodiment, at least one epithelial cancer biomarkerlevels are measured by MALDI-TOF mass spectrometry.

Antibodies

The antibodies for use in the present invention can be obtained from acommercial source. Alternatively, antibodies can be raised against theepithelial cancer biomarker polypeptide, or a portion of the epithelialcancer biomarker polypeptide.

Antibodies for use in the present invention can be produced usingstandard methods to produce antibodies, for example, by monoclonalantibody production (Campbell, A. M., Monoclonal Antibodies Technology:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, the Netherlands (1984); St. Groth et al.,J. Immunology, (1990) 35: 1-21; and Kozbor et al., Immunology Today(1983) 4:72). Antibodies can also be readily obtained by using antigenicportions of the protein to screen an antibody library, such as a phagedisplay library by methods well known in the art. For example, U.S. Pat.No. 5,702,892 (U.S.A. Health & Human Services) and WO 01/18058(Novopharm Biotech Inc.) disclose bacteriophage display libraries andselection methods for producing antibody binding domain fragments.

Detection Kit

The present invention is also directed to commercial kits for thedetection and prognostic evaluation of bladder cancer and for thediagnosis of invasive bladder cancer. The kit can be in anyconfiguration well known to those of ordinary skill in the art and isuseful for performing one or more of the methods described herein forthe detection of at least one epithelial cancer biomarker. The kits areconvenient in that they supply many if not all of the essential reagentsfor conducting an assay for the detection of at least one epithelialcancer biomarker in a biological sample. In addition, the assay ispreferably performed simultaneously with a standard or multiplestandards that are included in the kit, such as a predetermined amountof at least one epithelial cancer biomarker protein or nucleic acid, sothat the results of the test can be quantitated or validated.

The kits include a means for detecting at least one epithelial cancerbiomarker levels such as antibodies, or antibody fragments, whichselectively bind to at least one epithelial cancer biomarker protein.The diagnostic assay kit is preferentially formulated in a standardtwo-antibody binding format in which one at least one epithelial cancerbiomarker-specific antibody captures the biomarker in a patient sampleand another ADAM-specific antibody is used to detect captured at leastone epithelial cancer biomarker. For example, the capture antibody isimmobilized on a solid phase, e.g., an assay plate, an assay well, anitrocellulose membrane, a bead, a dipstick, or a component of anelution column. The second antibody, i.e., the detection antibody, istypically tagged with a detectable label such as a calorimetric agent orradioisotope.

In one preferred embodiment, the kit comprises a means for detectinglevels of at least one epithelial cancer biomarker in a sample of urine.In a specific embodiment, the kit comprises a “dipstick” with at leastone anti-epithelial cancer biomarker antibody or fragments, immobilizedthereon, which specifically bind a epithelial cancer biomarker protein.Specifically bound epithelial cancer biomarker protein can then bedetected using, for example, a second antibody that is detectablylabeled with a calorimetric agent or radioisotope.

In other embodiments, the assay kits may employ (but are not limited to)the following techniques: competitive and non-competitive assays,radioimmunoassay (RIA), bioluminescence and chemiluminescence assays,fluorometric assays, sandwich assays, immunoradiometric assays, dotblots, enzyme linked assays including ELISA, microtiter plates, andimmunocytochemistry. For each kit the range, sensitivity, precision,reliability, specificity and reproducibility of the assay areestablished by means well known to those skilled in the art.

The above described assay kits would further provide instructions foruse and a container to hold the urine sample.

All references cited above or below are herein incorporated byreference.

The present invention is further illustrated by the following Examples.

These Examples are provided to aid in the understanding of the inventionand are not construed as a limitation thereof.

Example 1 Proteomic Analysis of Voided Urine, Bladder Cancer Tissue andCell Lines for Biomarker Discovery in Transitional Cell CarcinomaIntroduction

There is a need for new biomarkers to aid in the diagnosis andmanagement of cancers of epithelial origin. Urine can serve as anexcellent medium for epithelial cancer biomarker discovery and analysis.Proteomic analysis by two-dimensional polyacrylamide gel electrophoresis(2D PAGE) is one effective tool to analyze the proteome of humanspecimens. We utilize 2D PAGE to analyze voided urine, human bladdertumor and normal tissue, and human derived bladder cancer cell lines asa method for biomarker discovery.

Methods Urine

Under IRB approved protocol, we collected voided urine specimens fromsixty-three patients prior to diagnostic cystoscopy with biopsy andtwenty-two age-matched control patients with no clinical evidence ofbladder cancer and no history of malignancy. Total urinary protein wasisolated and quantified. Equivalent amounts of protein from individualpatients were pooled into three groups: 1. State Ta, high grade; 2.Stage Ta, low grade; 3. Normal controls. Eight patients were included ineach group. A total of 40 ng of protein from each group (5 ng perpatient) were analyzed and compared by 2D PAGE.

Tissue

Under IRB approved protocol, bladder tumor tissue and normal urotheliumwere harvested from the cystectomy specimen of a patient with stage T3N1 M0 transitional cell carcinoma. Tissue specimens were immediatelyfrozen in liquid nitrogen and total protein was then isolated andquantified. 40 ng of protein from each tumor and normal tissue wereanalyzed and compared by 2D PAGE.

Cell Lines

Fractionated protein was isolated from two previously described celllines: 1. MGH-U1, cultured from high grade transitional cell carcinomaof the bladder and highly tumorigenic in nude mice; 2. MGH-U4, culturedfrom a patient with severe urothelial atypia and non-tumorigenic in nudemice. 40 ng of each cytoplasmic, nuclear and membrane protein fractionsfrom each cell line were analyzed and compared by 2D PAGe.

For all above specimens, unique protein spots were isolated and analyzedby liquid chromatography mass spectroscopy-mass spectroscopy (LCMS-MS).

Results

Analysis by 2D PAGE established a number of protein spots at commonmolecular weights (MW) and isoelectric points (pI) across the 3 groupsof urine specimens which represent the common or normal urinaryproteome. Similarly, we demonstrated common proteomic spectra for tissuespecimens and also for cell lines. The proteomic spectra of urine fromTa high grade patients, tumor tissue and MGH-U1 cell line revealedseveral similar peptide spots in the MW range 10-15 kD and pI 8-10 whichwere not present or have identified three of these proteins as CystatinB, an endogenous cysteine proteinase inhibitor, Chaperonin 10, a heatshock protein, and profilin, a cytoskeletal protein.

Conclusions

We demonstrate the discovery of three novel biomarkers for cancers ofepithelial origin.

Example 1I Immunostaining for Cystatin B in Bladder Cancer TissueMethods

Normal bladder and bladder cancer tissue were immunostained using mousemonoclonal anti-cystatin B antibody and counterstained withHaematoxylin. Immunostaining was performed using the bladder cancertissue microarray BL801 (US Biomax Inc, Rockville, Md.). The tissueswere deparaffinized, endogenous peroxide blocked in 3% hydrogen peroxidein methanol, and microwave antigen retrieval performed using AntigenUnmasking Solution. Blocking was performed using 5% normal horse serumand endogenous biotin blocked using Avidin/Biotin kit. Tissue wasincubated with mouse monoclonal anti-cystatin B/Stefin B antibody, cloneA6/2 (GeneTex, Inc, San Antonio, Tex.), followed by anti-mousebiotinylated secondary antibody, amplified using ABC kit, and developedusing DAB. Tissue was counterstained using Gill's Hematoxylin #3(Sigma-Aldrich, St. Louis, Mo.), and blued using Tacha's Bluing Solution(Biocare, Concord, Calif.). All reagents were purchased from VectorLaboratories, Burlingame, Calif., except where noted. All images werecaptured at equal exposure time.

Results

The levels of Cystatin B in samples from individuals with bladder cancerwere significantly higher than the levels observed in samples of normalbladder tissue.

1. A method for facilitating the diagnosis of a patient for a cancer ofepithelial origin comprising: a. obtaining a biological sample from thepatient; and b. detecting the presence or absence of at least oneepithelial cancer biomarker in the biological sample, wherein thepresence of at least one epithelial cancer biomarker is indicative ofcancer of epithelial origin, and wherein the epithelial cancer biomarkeris selected from the group consisting of Cystatin B, Chaperonin 10, andProfilin.
 2. A method for diagnosing a cancer of epithelial origin in apatient comprising: a. measuring at least one epithelial cancerbiomarker levels present in a biological sample obtained from thepatient, a test sample; b. comparing the level of at least oneepithelial cancer biomarker in the test sample with the level ofepithelial cancer biomarker present in a control sample; wherein ahigher level of at least one epithelial cancer biomarker in the testsample as compared to the level of epithelial cancer biomarker in thecontrol sample is indicative of cancer of epithelial origin, and whereinthe epithelial cancer biomarker is selected from the group consisting ofCystatin B, Chaperonin 10, and Profilin.
 3. The method of claim 1,wherein the cancer of epithelial origin is selected from the groupconsisting of breast cancer, basal cell carcinoma, adenocarcinoma,gastrointestinal cancer, lip cancer, mouth cancer, esophageal cancer,small bowel cancer, stomach cancer, colon cancer, liver cancer, bladdercancer, pancreas cancer, ovary cancer, cervical cancer, lung cancer,skin cancer, prostate cancer, and renal cell carcinoma. 4-6. (canceled)7. The method of claim 1, wherein the biological sample is urine.
 8. Themethod of claim 1, wherein the presence or absence of at least oneepithelial cancer biomarker or Cystatin B is detected using anantibody-based binding moiety which specifically binds to at least oneepithelial cancer biomarker or to Cystatin B.
 9. The method of claim 2,wherein the level of at least one epithelial cancer biomarker orCystatin B is measured by measuring the protein level of at least oneepithelial cancer biomarker protein or Cystatin B.
 10. The method ofclaim 9, wherein the protein level of epithelial cancer biomarker orlevel of Cystatin B is measured by a method comprising the steps of: a.contacting the test sample, or preparation thereof, with anantibody-based binding moiety which specifically binds the epithelialcancer biomarker or to Cystatin B to form an antibody-epithelial cancerbiomarker complex; and b. detecting the presence of the complex, therebymeasuring the level of epithelial cancer biomarker present.
 11. Themethod according to claim 8, wherein the antibody-based binding moietyis labeled with a detectable label.
 12. The method according to claim11, wherein the label is selected from the group consisting of aradioactive label, a hapten label, a fluorescent label, and an enzymaticlabel.
 13. The method according to claim 8, wherein the antibody-basedbinding moiety is an antibody.
 14. The method according to claim 13,wherein the antibody is an monoclonal antibody. 15-19. (canceled) 20.The method of claim 2, wherein the cancer of epithelial origin isselected from the group consisting of breast cancer, basal cellcarcinoma, adenocarcinoma, gastrointestinal cancer, lip cancer, mouthcancer, esophageal cancer, small bowel cancer, stomach cancer, coloncancer, liver cancer, bladder cancer, pancreas cancer, ovary cancer,cervical cancer, lung cancer, skin cancer, prostate cancer, and renalcell carcinoma.
 21. The method according to claim 10, wherein theantibody-based binding moiety is labeled with a detectable label. 22.The method according to claim 10, wherein the antibody-based bindingmoiety is an antibody.